Uridine nucleotides as ganglionic neurotransmitters: characterization of presynaptic release and identification of postsynaptic receptors in rat superior cervical ganglia

Postganglionic neurons of rat superior cervical ganglia (SCG) possess at least two types of excitatory nucleotide receptors: an ATP-sensitive cation channel (a P2X purinoceptor), and a receptor activated by uridine nucleotides. In previous studies, we identified these receptors by showing that their activation triggers Ca2+-dependent and action potential-mediated noradrenaline release. Nevertheless, several important questions about the roles of nucleotides and their receptors in the SCG remained open. These questions refer (a) to the release of endogenous adenine and uridine nucleotides from presynaptic nerve terrninals in SCG, (b) to the nature of the receptor activated by uridine nucleotides, and (c) to the intracellular signalling cascade of this uridine nucleotide receptor. Ad (a): Exocytotic release of ATP in the SCG has been demonstrated recently, but evidence for neuronal release of UTP is lacking. A novel enzymatic method will be employed to determine whether stimulation of preganglionic axons leads to the liberation of UTP in the ganglion. This would establish uridine nucleotides as a novel class of ganglionic transmitters. Ad (b): In preliminary experiments, UTP reduced steady outward currents of rat SCG neurons which arise at potentials positive to -60 mV, an effect mimicked by the muscarinic agonist oxotremorine M. Recordings of outward currents under conditions which favour the isolation of transient (A) currents, delayed rectifier currents, fast and slow Ca2+-dependent K+ currents, and of muscarinic K+ (M) currents confirmed that UTP selectively inhibited muscarinic K+ channels. This action of UTP was half maximal at 2 M and was attenuated by 30 M PPADS but not by suramin. UDP was an equipotent agonist, while ADP was tenfold less potent, and ATP was inactive. This pharmacological profile is not compatible with any of the metabotropic nucleotide (P2Y) receptors characterized by molecular cloning. Experiments under conditions that minimize metabolism of nucleotides and with the use of additional antagonists will be performed to further characterize this receptor. Moreover, RNA extraction followed by reverse transcription, PCR amplification, and intracellular injection of corresponding antisense RNA will be employed to unequivocally identify the uridine nucleotide receptors mediating the inhibition of M-type K+ channels. A detailed description of receptors mediating effects of endogeneously released nucleotides might provide novel sites of action for future pharmacotherapies. Ad (c): Receptors for uridine nucleotides belong to the superfamily of G protein coupled receptors. Accordingly, the UTP inhibition of M currents was abolished when intracellular GTP was replaced by GDPb S and became irreversible when GTP was replaced by GTPg S. Dialysis of neurons with fast Ca2+ buffers also attenuated the UTP-induced inhibition of M currents which points to a role of receptor-dependent increases of intracellular Ca2+, an UTP effect that will be confirmed in this project. In addition, UDP and UTP raised the levels of cellular inositol triphosphates (IP3 ). Hence, uridine nucleotide receptors of rat SCG neurons, like all the cloned P2Y receptors investigated in heterologous expression systems, obviously activate phospholipase C to generate IP 3 . Future experiments will clarify which G protein a subunits are involved in the action of UTP and whether it is the a or the b g subunits mediating the effect. Furthermore, IP 3 , cyclic GMP, cyclic ADP ribose, and ryanodine receptors will be investigated as potential signalling molecules involved in the action of uridine nucleotide receptors. Results from these experiments might reveal novel signalling mechanisms of metabotropic receptors in neurons.

The data obtained within the project 12997 characterize 3 different receptors for extracellular nucleotides, which all are involved in signalling within the sympathetic nervous system. In future, agents that selectively activate or block one or more of these receptors, could be used as therapeutic agents for malfunctions of the sympathetic nervous system, such as hypertension. Within the last 10 years, ATP has been firmly established as a neurotransmitter in the central as well as peripheral nervous system. During the same time, seven ATP-gated ion channels (P2X) and as many G protein-coupled (P2Y) receptors for nucleotides have been described by molecular techniques. Some of these receptors recognize not only adenine, but also uridine nucleotides as agonists. In previous experiments, we have found out that postganglionic sympathetic neurons within superior cervical ganglia of rats possess endogenous receptors for adenine as well as uridine nucleotides. Activation of both of these receptors depolarizes the neurons and thus triggers transmitter release. The data obtained now during the course of project 12997 characterize the receptors involved, as well as the underlying intracellular signalling mechanisms. The major results can be summarized as follows: (1) Sympathetic neurons express presynaptic P2X receptors which mediate positive feedback modulation of sympatho-effector transmission (Boehm, 1999). (2) Sympathetic neurons express presynaptic P2Y receptors which mediate negative feedback modulation of sympatho-effector transmission (Boehm, 1999). These receptors may correspond to the recently cloned P2Y 12 subtype (Vartian and Boehm, 2001). (3) SCG neurons express somatic P2Y6 receptors which may be involved in ganglionic transmission (Boehm et al, 1995; Boehm, 1998; Boehm, 1999; Vartian et al, 2001). (4) The P2Y6 receptors are linked to two different signalling cascades: via Gq-like G proteins phospholipase C is activated, and an inositoltrisphosphate-dependent liberation of intracellular Ca2+ causes inhibition of M-type K+ channels (Bofill-Cardona et al, 2000); via Gi-like G proteins, proteinkinase C is activated and elicits action potential-mediated transmitter release (Vartian et al, 2001). Taken together, these results provide insight into the roles of nucleotides and their receptors in the sympathetic nervous system. (5) In addition, results concerning the presynaptic modulation of transmitter release have been obtained (Boehm and Huck, 1998; Boehm, 1999), and these preliminary data have been used to apply for additional scientific grants provided by the FWF. The two grant applications submitted (P13920 and P14951) have been successful. Thus, the present project will have impact on future research. (6) Finally, the cognition enhnacer linopirdine was demonstrated to augment transmitter release through its action on M-type K+ channels (Kristufek et al, 1999). This provides new insights into neuronal mechanisms that may be important in the treatment of Alzheimers disease.